The field of the present invention is imaging using a light guide bundle.
Microscopes magnify objects or samples, which can be stationary and moving. One type of microscope is a confocal microscope, which uses a very small spot, or pinhole, of light to make its image of the target. Typically, the spot is scanned across the target in a pointwise, digital fashion and the image is made by combining the points of return light emanating from the target (the return light can be, for example, reflected light, fluorescent light, or an exotic form of light such as a Raman spectrum, and can be found in any desirable region of the electromagnetic spectrum, such as ultraviolet (UV) light, blue light, visible light, near-infrared (NIR) light and infrared (IR) light).
The confocal geometry of the illumination pinhole, the object, and the detection pinhole give a higher resolution image than a conventional widefield microscope. In some embodiments, confocal microscopy can improve the spatial resolution about 1.3 times. See, e.g., U.S. Pat. No. 5,587,832. Confocal microscopy also improves the xe2x80x9cup and downxe2x80x9d (i.e., z-axis or axial) resolution, which gives rise to an extremely useful optical sectioning capability, which means that images can be obtained at different depths, and thus 3-D images and volume reconstruction can be obtained.
In order to obtain the pointwise image, confocal microscopes can either move a specimen and keep the optics fixed in place, or they can keep the specimen fixed and move the light beam, for example by scanning the beam using special rotating aperture disks or other beam scanners. See U.S. Pat. Nos. 4,802,748, 5,067,805, 5,099,363, 5,162,941. Other confocal scanning systems have used a laser beam rastered with rotating mirrors to scan a specimen or a laser beam that scans a slit rather than a spot; such slit scanning increases imaging speed but slightly degrades resolution. See U.S. Pat. No. 5,587,832.
Confocal microscopes typically use a bulky design in which several large componentsxe2x80x94including a laser system as the light source, detection pinholes, x-y beam steering devices, and an optical detectorxe2x80x94must be carefully maintained in precise alignment. In these systems, the specimen or target to be imaged is placed on a stage as in a conventional microscope. These limitations make the confocal microscope cumbersome, inflexible and inconvenient for imaging specimens which are not easily accessible or easily placed on a microscope stage. In other words, present confocal systems are designed for in vitro imaging of biological specimens in the lab instead of imaging tissues in the body, in vivo.
Several approaches have been proposed to permit in vivo imaging. See, e.g., T. Dabbs and M. Glass, xe2x80x9cFiber-optic confocal microscope: FOCON,xe2x80x9d Applied Optics, vol. 31, pp. 3030-3035, 1992; L. Giniunas, R. Juskatis, and S. V. Shatalin, xe2x80x9cScanning fiber-optic microscope,xe2x80x9d Electronic Letters, vol. 27, pp. 724-725, 1991; L. Giniunas, R. Juskatis, and S. V. Shatalin, xe2x80x9cEndoscope with optical sectioning capability,xe2x80x9d Applied Optics, vol. 32, pp. 2888-2890, 1993; D. L. Dickensheets and G. S. Kino, xe2x80x9cMicromachined scanning confocal optical microscope,xe2x80x9d Optics Letters, vol. 21, pp. 764-766, 1996; D. L. Dickensheets and G. S. Kino, xe2x80x9cMiniature scanning confocal microscope,xe2x80x9d U.S. Pat. No. 5,907,425 (continuation of U.S. Pat. No. 5,742,419), May 1999; A. F. Gmitro and D. Aziz, xe2x80x9cConfocal microscopy through a fiber-optic imaging bundle,xe2x80x9d Optics Letters, vol. 18, pp. 565-567, 1993; Y. S. Sabharwal, A. R. Rouse, L. Donaldson, M. F. Hopkins, and A. F. Gmitro, xe2x80x9cSlit-scanning confocal microendoscope for high-resolution in vivo imaging, Applied Optics, vol. 38, pp. 7133-7144, 1999; R. Juskaitis, T. Wilson, and T. F. Watson, xe2x80x9cConfocal microscopy using optical fibre imaging bundles,xe2x80x9d Proceedings of SPIE, vol. 2655, pp. 92-94, 1996; U.S. Pat. No. 5,587,832; PCT/CA98/00993, Publication No. WO 99/22262. None of these systems provide as high a quality of image as could be desired for various aspects of microscopy.
Thus, there has gone unmet a need for improved microscopy systems, including confocal microscopy systems, wherein the systems can provide high quality images of desired targets in locations where the positioning of the target might not be carefully controlled, including in vivo targets. The present invention provides these and other advantages.
The present invention comprises microscopes and methods that have significant advantages in controlling the light that contacts a sample and/or that is detected emanating from a sample. The microscopes and methods, which preferably relate to confocal microscopes and further preferably confocal endoscopes for in vivo imaging, comprise a spatial light modulator in the illumination and/or detection light path so that light transmitted to the target, for example via a bundle of light guides, is transmitted substantially only into the cores of the light guide bundle and not into inter-core areas such as the cladding surrounding the light guides or filler between the light guides in the bundle. This may reduce the amount of noise or stray light in the image from the target tissue, thereby enhancing the sensitivity, contrast or resolution of the image, in at least one of the x-y directions and in the z-direction, and provides other related advantages. The present invention may also provide systems comprising only a single light guide bundle in a microendoscope and can reduce cross-talk between light guides.
In one aspect, the present invention provides a viewing system comprising a spatial light modulator and a light guide bundle having a proximal end and a distal end, wherein spatial light modulator is optically connected to the proximal end of the light guide bundle in a same conjugate image plane as the proximal end such that the spatial light modulator controls the location of light impinging on the proximal end. In some embodiments, the viewing system of comprises an endoscope or the light guide bundle comprises at least 100 light guides. The endoscope can be a confocal microscopy endoscope. The spatial light modulator can be operably connected to a controller comprising computer-implemented programming able to set to an on-state pixels of the spatial light modulator corresponding to cores of corresponding light guides in the light guide bundle to provide on-pixels and able to set to an off-state pixels corresponding to inter-core areas of the light guide bundle to provide off-pixels.
In other embodiments, a plurality of selected groups of the on-pixels are in the on-state, the selected groups being spaced apart such that light emanating from the distal end of a first light guide corresponding to a first selected group of on-pixels does not substantially interfere with light emanating from the distal end of a second light guide corresponding to a second selected group of on-pixels, and substantially all other pixels of the spatial light modulator are in the off-state. Typically, at least 3 different pixels of the spatial light modulator correspond to each core of substantially all of the corresponding light guides. The viewing system can further comprise a pixelated detector optically connected to receive light emanating from the proximal end of the light guide bundle and the controller further comprises computer-implemented programming that distinguishes between light emanating from the light guides corresponding to on-pixels of the spatial light modulator and light emanating from other light guides. The computer-implemented programming can additionally ignores light emanating from the other light guides.
In further embodiments, the controller further comprises computer-implemented programming that detects light emanating from the other light guides to provide out-of-focus data and the programming incorporates the out-of-focus data with the light emanating from the light guides corresponding to the on-pixels to provide an enhanced image. The out-of-focus data can be fit using the light emanating from the light guides corresponding to the on-pixels using a 2D Gaussian distribution or using desired point spread functions as described herein.
The viewing system can be a single-pass or double-pass viewing system, and the viewing system can further comprise a light source optically connected to the proximal end of the light guide bundle and the spatial light modulator is optically connected between the light source and the proximal end of the light guide bundle. Where the viewing system is a double-pass viewing system, and the viewing system can further comprise a light source and a detector that are both optically connected to the proximal end of the light guide bundle, and the spatial light modulator is optically connected between a) each of the light source and the detector, and b) the proximal end of the light guide bundle. In some embodiments, the controller further comprises computer-implemented programming that maps pixels of the spatial light modulator to corresponding cores of corresponding light guides in the light guide bundle to provide a map comprising corresponding pixels and non-corresponding pixels.
The viewing system can further comprise a scanner that controls the location of light transmitted to the spatial light modulator and on to the proximal end of the light guide bundle, and the controller further comprises computer-implemented programming that directs the scanner to scan the spatial light modulator and simultaneously sets at least one of the corresponding pixels to an on-state and sets other pixels of the spatial light modulator to an off-state, thereby causing light from the light source to be transmitted substantially only to the cores of corresponding light guides. The viewing system can also comprise a light source optically connected to the spatial light modulator such that the light source illuminates a substantial portion of the pixels of the spatial light modulator, and the controller further comprises computer-implemented programming that sets selected corresponding pixels to an on-state and sets other pixels of the spatial light modulator to an off-state, thereby causing light from the light source to be transmitted substantially only to the cores of the light guides corresponding to the corresponding pixels. The controller can further comprise computer-implemented programming that selects the selected corresponding pixels that are set to an on-state such that light emanating from the distal end of a first light guide corresponding to a first selected corresponding pixel does not substantially interfere with light emanating from the distal end of a second light guide corresponding to a second selected corresponding pixel, and the selected corresponding pixels that are set to an on-state are varied over time such that substantially all of the light guides in the light guide bundle are illuminated.
In another aspect, the present invention provides a flexible endoscope system providing confocal microscopy of a target tissue, the system comprising an endoscope comprising a light guide bundle comprising at least 100 light guides and having a proximal end and a distal end, the system further comprising a spatial light modulator that is optically connected to the proximal end of the light guide bundle in a same conjugate image plane as the proximal end such that the spatial light modulator controls the location of light impinging on the proximal end, and a controller comprising computer-implemented programming that is operably connected to the spatial light modulator and that is able to set to an on-state groups of pixels of the spatial light modulator corresponding to cores of corresponding light guides in the light guide bundle to provide groups of on-pixels and able to set to an off-state pixels corresponding to inter-core areas of the light guide bundle to provide off-pixels. A plurality of selected groups of the on-pixels can be in the on-state, the selected groups being spaced apart such that light emanating from the distal end of a first light guide corresponding to a first selected group of on-pixels does not substantially interfere with light emanating from the distal end of a second light guide corresponding to a second selected group of on-pixels, and other pixels of the spatial light modulator are in the off-state.
The endoscope can further comprise a pixelated detector optically connected to receive light emanating from the proximal end of the light guide bundle and the controller further comprises computer-implemented programming that distinguishes between light emanating from the light guides corresponding to on-pixels of the spatial light modulator and light emanating from other light guides.
The present invention also provides methods of making an viewing system comprising: a) providing a spatial light modulator; b) providing a light guide bundle having a proximal end and a distal end; and, c) placing the spatial light modulator in optical connection to the proximal end of the light guide bundle in a same conjugate image plane as the proximal end such that the spatial light modulator controls the location of light impinging on the proximal end. The viewing system can be a confocal microscopy endoscope and the method further comprises providing the light guide bundle comprising at least 100 light guides. The methods can further comprise operably connecting the spatial light modulator to a controller comprising computer-implemented programming able to set to an on-state pixels of the spatial light modulator corresponding to cores of corresponding light guides in the light guide bundle to provide on-pixels and able to set to an off-state pixels; corresponding to inter-core areas of the light guide bundle to provide off-pixels.
The methods can further comprise optically connecting a pixelated detector to the system to receive light emanating from the proximal end of the light guide bundle and further providing the controller with computer-implemented programming that distinguishes between light emanating from the light guides corresponding to on-pixels of the spatial light modulator and light emanating from other light guides. The method can be directed to making a single-pass or double-pass viewing system, and can further comprise providing a scanner that controls the location of light transmitted to the spatial light modulator and on to the proximal end of the light guide bundle or optically connecting the light source to the spatial light modulator such that the light source illuminates a substantial portion of the pixels of the spatial light modulator.
The present invention further provides methods of making a flexible endoscope system comprising: a) providing a spatial light modulator; b) providing a light guide bundle comprising at least 100 light guides having a proximal end and a distal end, at least the distal end of the light guide bundle disposed within an endoscope; c) placing the spatial light modulator in optical connection to the proximal end of the light guide bundle in a same conjugate image plane as the proximal end such that the spatial light modulator controls the location of light impinging on the proximal end; and, d) operably connecting a controller comprising computer-implemented programming to the spatial light modulator wherein the controller is able to set to an on-state groups of pixels of the spatial light modulator corresponding to cores of corresponding light guides in the light guide bundle to provide groups of on-pixels and able to set to an off-state pixels corresponding to inter-core areas of the light guide bundle to provide off-pixels. Such methods can further comprise optically connecting a pixelated detector to the system to receive light emanating from the proximal end of the light guide bundle and further providing the controller with computer-implemented programming that distinguishes between light emanating from the light guides corresponding to on-pixels of the spatial light modulator and light emanating from other light guides.
The present invention still further provides methods of illuminating a target comprising: a) transmitting light from a light source to a proximal end of a light guide bundle via a spatial light modulator wherein the spatial light modulator transmits the light substantially only to cores of light guides in the light guide bundle; b) transmitting the light from the proximal end of the light guide bundle to a distal end of the light guide bundle and emitting the light from the distal end of the light guide bundle; and, c) illuminating the target with the light emitted from the distal end of the light guide bundle. The methods can comprise scanning a light beam across the spatial light modulator and simultaneously setting at least one pixel of the spatial light modulator that corresponds to a core of one of the light guides to an on-state to provide at least one on-pixel and setting other pixels of the spatial light modulator to an off-state, whereby the light beam is transmitted substantially only to the core of the light guide when the light beam contacts the on-pixel and the light beam is not transmitted to inter-core areas of the light guide bundle or to light guides adjacent to the light guide. The light beam can be a laser beam or other desired light beam.
In some embodiments, the methods comprise scanning the light beam across substantially all pixels that are set to an on-state over time such that substantially all of the light guides in the light guide bundle are illuminated, thereby illuminating substantially all of the target within a field of view of the light guide bundle without moving the light guide bundle. In further embodiments, the methods comprise optically connecting the light source to the spatial light modulator such that the light source illuminates a substantial portion of the pixels of the spatial light modulator, and setting selected corresponding pixels to an on-state and setting other pixels of the spatial light modulator to an off-state such that light from the light source is transmitted substantially only to the cores of the light guides corresponding to the corresponding pixels. The method can comprise varying the selected corresponding pixels that are set to an on-state over time such that substantially all of the light guides in the light guide bundle are illuminated, thereby illuminating substantially all of the target within a field of view of the light guide bundle without moving the light guide bundle.
The methods can comprise selecting the selected corresponding pixels that are set to an on-state such that light emanating from the distal end of a first light guide corresponding to a first selected corresponding pixel does not substantially interfere with light emanating from the distal end of a second light guide corresponding to a second selected corresponding pixel.
The present invention also provides methods of obtaining an image of a target comprising: a) transmitting light from a light source via a spatial light modulator to a light guide bundle, then emitting the light from a distal end of the light guide bundle to illuminate the target and thereby cause light to emanate from the target to provide emanating light; b) collecting the emanating light that contacts the distal end of the light guide bundle; and c) transmitting the emanating light via the light guide bundle to a detector to provide an image of the target at the detector. The detector can comprise, for example, an eyepiece ocular or a pixelated detector, and the image can be a confocal image.
The methods can comprise setting to an on-state pixels of the spatial light modulator that correspond to cores of corresponding light guides in the light guide bundle to provide on-pixels and setting to an off-state pixels corresponding to inter-core areas of the light guide bundle to provide off-pixels. The methods can also comprise setting a plurality of selected groups of the on-pixels to an on-state wherein the selected groups are spaced apart such that light emanating from the distal end of a first light guide corresponding to a first selected group of on-pixels does not substantially interfere in the target with light emanating from the distal end of at least one second light guide corresponding to at least one second selected group of on-pixels, and substantially all other pixels of the spatial light modulator are in the off-state. The methods can further comprise distinguishing between light emanating from the light guides corresponding to on-pixels of the spatial light modulator and light emanating from other light guides, then ignoring light emanating from the other light guides or evaluating the light emanating from the other light guides to provide out-of-focus data and the incorporating the out-of-focus data with the light emanating from the light guides corresponding to the on-pixels to provide an enhanced image.
The methods can be effected using a single-pass viewing system such that the spatial light modulator acts as an illumination mask such that illumination light is transmitted substantially only to light guide cores of light guides that correspond to on-pixels of the spatial light modulator, or a double-pass viewing system, such that the spatial light modulator acts as an illumination mask such that illumination light is transmitted substantially only to corresponding light guides and as a detection mask that substantially prevents light from light guides other than corresponding light guides from reaching the detector.
The methods can comprise mapping pixels of the spatial light modulator to corresponding cores of corresponding light guides in the light guide bundle to provide a map comprising corresponding pixels and non-corresponding pixels.
These and other aspects, features and embodiments are set forth within this application, including the following Detailed Description and attached drawings. In addition, various references are set forth herein, including in the Cross-Reference To Related Applications, that describe in more detail certain compositions, apparatus, methods and other information (e.g., spatial light modulators, etc.); all such references are incorporated herein by reference in their entirety and for all their teachings and disclosures, regardless of where the references may appear in this application.